Tumors actively suppress immune response against themselves. This creates a fundamental barrier to successful immunotherapy, but the underlying molecular mechanisms are still poorly understood. This proposal builds upon the novel discovery that three key molecular pathways - indoleamine 2,3-dioxygenase (IDO), CD40/CD40L and CTLA-4 - all become tightly linked in tumor-bearing hosts, functioning together as a single integrated regulatory network. This innovative model is supported by important new discoveries made during the previous period of support, including the role of IDO in activating highly suppressive Tregs in tumor-bearing hosts; and identification of a novel subset of reprogrammable Foxp3+ Tregs that is capable of converting into pro-inflammatory, CD40L-expressing helper T cells under suitable conditions. The current proposal will use informative mouse preclinical models to develop clinically-applicable, mechanistically-based immunotherapy regimens. These regimens will incorporate the first-in human IDO-inhibitor drug 1-methyl-D-tryptophan (1MT), now in Phase I clinical trials, in combination with chemotherapy and active immunotherapy. Aim 1 will test the hypothesis that the highly suppressive Tregs in tumor-draining lymph nodes (TDLNs) can be de-activated and rendered non-suppressive by simultaneously blocking IDO and providing a strong proinflammatory signal through the CD40/CD40L pathway. Aim 2 will test the hypothesis that DCs in TDLNs can be licensed for robust and effective cross-presentation of endogenous tumor antigens by preventing the tolerogenic DC phenotype that is induced by IDO-activated Tregs via the CTLA-4/B7/FOXO3 pathway, and simultaneously driving the DC-activating/licensing CD40/CD40L pathway. Aim 3 will test the hypothesis that the late stages of the CD8+ cytotoxic T cell response (clonal expansion, effector differentiation, and long-term memory formation) can be markedly enhanced by blocking the cell-intrinsic inhibitory effect of CTLA-4 on the CD8+ cells, and that this cell-intrinsic effect of CTLA-4 different from - and acts synergistically with - the upstream cell-extrinsic effects of interrupting the IDO/CTLA-4 loop during the initial priming stage. These Specific Aims continue the project's strongly translational and pre-clinical therapeutic focus. The overall goal of the current proposal is to develop novel high-potency combination regimens that leverage the synergy of IDO-inhibitor drugs in combination with CD40 agonist antibodies, chemotherapy, and CTLA-4 blockade. The strategies proposed are timely, high-impact, and have a clear path to the clinic. The proposed studies bring new mechanistic insights to bear on the difficult and important clinical problem of identifying highly potent and synergistic multi-aget immunotherapy regimens, and provide testable outcome measures at the molecular level to evaluate whether each proposed strategic intervention is hitting its hypothesized target.